Genetic loss of D-amino acid oxidase activity reverses schizophrenia-like phenotypes in mice

Reduced function of the N -methyl- d -aspartate receptor (NMDAR) has been implicated in the pathophysiology of schizophrenia. The NMDAR contains a glycine binding site in its NR1 subunit that may be a useful target for the treatment of schizophrenia. In this study, we assessed the therapeutic potential of long-term increases in the brain levels of the endogenous NMDAR glycine site agonist D-serine, through the genetic inactivation of its catabolic enzyme D-amino acid oxidase (DAO) in mice. The effects of eliminating DAO function were investigated in mice that display schizophrenia-related behavioral deficits due to a mutation ( Grin 1 ^D481N ) in the NR1 subunit that results in a reduction in NMDAR glycine affinity. Grin 1 ^D481N mice show deficits in sociability, prolonged latent inhibition, enhanced startle reactivity and impaired spatial memory. The hypofunctional Dao 1 ^G181R mutation elevated brain levels of D-serine, but alone it did not affect performance in the behavioral measures. Compared to animals with only the Grin 1 ^D481N mutation, mice with both the Dao1 ^G181R and Grin 1 ^D481N mutations displayed an improvement in social approach and spatial memory retention, as well as a reversal of abnormally persistent latent inhibition and a partial normalization of startle responses. Thus, an increased level of D-serine resulting from decreased catalysis corrected the performance of mice with deficient NMDAR glycine site activation in behavioral tasks relevant to the negative and cognitive symptoms of schizophrenia. Diminished DAO activity and elevations in D-serine may serve as an effective therapeutic intervention for the treatment of psychiatric symptoms.     

Protein kinase C activity regulates D-serine availability in the brain

D-serine is a co-agonist of NMDA receptor (NMDAR) and plays important roles in synaptic plasticity mechanisms. Serine racemase (SR) is a brain-enriched enzyme that converts L-serine to D-serine. SR interacts with the protein interacting with C-kinase 1 (PICK1), which is known to direct protein kinase C (PKC) to its targets in cells. Here, we investigated whether PKC activity regulates SR activity and D-serine availability in the brain. In vitro, PKC phosphorylated SR and decreased its activity. PKC activation increased SR phosphorylation in serine residues and reduced D-serine levels in astrocyte and neuronal cultures. Conversely, PKC inhibition decreased basal SR phosphorylation and increased cellular D-serine levels. In vivo modulation of PKC activity regulated both SR phosphorylation and D-serine levels in rat frontal cortex. Finally, rats that completed an object recognition task showed decreased SR phosphorylation and increased D-serine/total serine ratios, which was markedly correlated with decreased PKC activity in both cortex and hippocampus. Results indicate that PKC phosphorylates SR in serine residues and regulates D-serine availability in the brain. This interaction may be relevant for the regulation of physiological and pathological mechanisms linked to NMDAR function.     

Potential role for astroglial D-amino acid oxidase in extracellular D-serine metabolism and cytotoxicity

D-amino acid oxidase (DAO) is a flavoenzyme that catalyzes the oxidation of D-amino acids. In the brain, gene expression of DAO is detected in astrocytes. Among the possible substrates of DAO in vivo, D-serine is proposed to be a neuromodulator of the N-methyl-D-aspartate (NMDA) receptor. In a search for the physiological role of DAO in the brain, we investigated the metabolism of extracellular D-serine in glial cells. Here we show that after D-serine treatment, rat primary type-1 astrocytes exhibited increased cell death. In order to enhance the enzyme activity of DAO in cells, we established stable rat C6 glial cells overexpressing mouse DAO designated as C6/DAO. Treatment with a high dose of D-serine led to the production of hydrogen peroxide (H(2)O(2)) followed by apoptosis in C6/DAO cells. Among the amino acids tested, D-serine specifically exhibited a significant cell death-inducing effect. DAO inhibitors, i.e., sodium benzoate and chlorpromazine, partially prevented the death of C6/DAO cells treated with D-serine, indicating the involvement of DAO activity in d-serine metabolism. Overall, we consider that extracellular D-serine can gain access to intracellular DAO, being metabolized to produce H(2)O(2). These results support the proposal that astroglial DAO plays an important role in metabolizing a neuromodulator, D-serine.     

Crystal structure of human D-amino acid oxidase: context-dependent variability of the backbone conformation of the VAAGL hydrophobic stretch located at the si-face of the flavin ring

In the brain, the extensively studied FAD-dependent enzyme D-amino acid oxidase (DAO) degrades the gliotransmitter D-serine, a potent activator of N-methyl-D-aspartate type glutamate receptors, and evidence suggests that DAO, together with its activator G72 protein, may play a key role in the pathophysiology of schizophrenia. Indeed, its potential clinical importance highlights the need for structural and functional analyses of human DAO. We recently succeeded in purifying human DAO, and found that it weakly binds FAD and shows a significant slower rate of flavin reduction compared with porcine DAO. However, the molecular basis for the different kinetic features remains unclear because the active site of human DAO was considered to be virtually identical to that of porcine DAO, as would be expected from the 85% sequence identity. To address this issue, we determined the crystal structure of human DAO in complex with a competitive inhibitor benzoate, at a resolution of 2.5 Angstrom. The overall dimeric structure of human DAO is similar to porcine DAO, and the catalytic residues are fully conserved at the re-face of the flavin ring. However, at the si-face of the flavin ring, despite the strict sequence identity, a hydrophobic stretch (residues 47-51, VAAGL) exists in a significantly different conformation compared with both of the independently determined porcine DAO-benzoate structures. This suggests that a context-dependent conformational variability of the hydrophobic stretch accounts for the low affinity for FAD as well as the slower rate of flavin reduction, thus highlighting the unique features of the human enzyme.     

Structural basis of D-DOPA oxidation by D-amino acid oxidase: alternative pathway for dopamine biosynthesis

D-amino acid oxidase (DAO) degrades the gliotransmitter D-serine, a potent endogenous ligand of N-methyl-D-aspartate type glutamate receptors. It also has been suggested that D-DOPA, the stereoisomer of L-DOPA, is oxidized by DAO and then converted to dopamine via an alternative biosynthetic pathway. Here, we provide direct crystallographic evidence that D-DOPA is readily fitted into the active site of human DAO, where it is oxidized by the enzyme. Moreover, our kinetic data show that the maximal velocity for oxidation of D-DOPA is much greater than for D-serine, which strongly supports the proposed alternative pathway for dopamine biosynthesis in the treatment of Parkinson's disease. In addition, determination of the structures of human DAO in various states revealed that the conformation of the hydrophobic VAAGL stretch (residues 47-51) to be uniquely stable in the human enzyme, which provides a structural basis for the unique kinetic features of human DAO.     

D-serine: a new word in the glutamatergic neuro-glial language

Summary. Gliotransmission is a process in which astrocytes are dynamic elements that influence synaptic transmission and synaptogenesis. The best-known gliotransmitters are glutamate and ATP. However, in the past decade, it has been demonstrated that D-serine, a D-amino acid, acts as a gliotransmitter in glutamatergic synapses. The physiological relevance of D-serine is sustained by the way in which it modulates the action of glutamatergic neurotransmission, neuronal migration and long-term potentiation (LTP). In addition, the synthesis and degradation mechanisms of D-serine have been proposed as potential therapeutic targets for the treatment of Alzheimer’s disease, schizophrenia and related disorders. In the present review, detailed information is provided about the physiological and physiopathological relevance of D-serine, including metabolic and regulation aspects.     

D构象丝氨酸在中枢神经系统中的功能研究进展

哺乳动物中枢神经系统中D构象丝氨酸的区域性高浓度分布与N-甲基-D-天冬氨酸(NMDA)受体相一致.它主要由丝氨酸消旋酶将L丝氨酸直接消旋而来,也可能通过肠道菌群产生后吸收至体内,最终被D构象氨基酸氧化酶氧化.这种从胶质细胞而非神经元来源的“异常”构象氨基酸作为一种新型神经递质,不仅更新了传统“神经递质”的定义,而且为许多与NMDA受体过度兴奋或表达下调相关的神经系统疾病治疗提出了新的线索.     

AMPA receptor mediated D-serine release from retinal glial cells

The N-methyl-D-aspartate receptor (NMDAR) co-agonist D-serine is important in a number of different processes in the CNS, ranging from synaptic plasticity to disease states, including schizophrenia. D-serine appears to be the major co-agonist acting on retinal ganglion cell NMDA receptors, but the cell type from which it originates and whether its release can be modulated by activity are unknown. In this study, we utilized a mutant mouse line with elevated d-serine to investigate this question. Direct measurements of extracellular D-serine using capillary electrophoresis demonstrate that D-serine can be released from the intact mouse retina through an α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPAR) dependent mechanism. α-Amino-3-hydroxyl-5-methyl-4-isoxazole-propionate-evoked D-serine release persisted in the presence of a cocktail of neural inhibitors but was abolished after administration of a glial toxin. These findings provide the first evidence that extracellular D-serine levels in the retina can be modulated, and that such modulation is contingent upon glial cell activity.     

Immunochemical relationship of D-amino acid oxidases in various tissues and animals.

1. By means of an enzyme immunoassay, the contents of D-amino acid oxidase (DAO) were determined in kidney, liver, cerebellum and lung of hog, but the oxidase was not detectable in heart or cerebrum. 2. The oxidases in kidney, liver and cerebellum of hog were indistinguishable as regards immunoreactivity toward anti-hog kidney DAO antibody, specific activity and molecular weight. 3. The oxidases in rat and dog kidneys immunochemically cross-reacted with anti-hog DAO antibody. 4. The overall structure of the hog oxidase was more similar to that of the dog enzyme than that of the rat, while the structure around the catalytic site of the hog oxidase was more similar to that of the rat oxidase than that of the dog enzyme. 5. On immunoblot analysis, two forms of the oxidase were detected in extracts of hog, rat and dog kidneys.     

Paradoxical roles of serine racemase and D-serine in the G93A mSOD1 mouse model of amyotrophic lateral sclerosis

D-serine is an endogenous neurotransmitter that binds to the NMDA receptor, thereby increasing the affinity for glutamate, and the potential for excitotoxicity. The primary source of D-serine in vivo is enzymatic racemization by serine racemase (SR). Regulation of D-serine in vivo is poorly understood, but is thought to involve a combination of controlled production, synaptic reuptake by transporters, and intracellular degradation by D-amino acid oxidase (DAO). However, SR itself possesses a well-characterized eliminase activity, which effectively degrades D-serine as well. D-serine is increased two-fold in spinal cords of G93A Cu,Zn-superoxide dismutase (SOD1) mice--the standard model of amyotrophic lateral sclerosis (ALS). ALS mice with SR disruption show earlier symptom onset, but survive longer (progression phase is slowed), in an SR-dependent manner. Paradoxically, administration of D-serine to ALS mice dramatically lowers cord levels of D-serine, leading to changes in the onset and survival very similar to SR deletion. D-serine treatment also increases cord levels of the alanine-serine-cysteine transporter 1 (Asc-1). Although the mechanism by which SOD1 mutations increases D-serine is not known, these results strongly suggest that SR and D-serine are fundamentally involved in both the pre-symptomatic and progression phases of disease, and offer a direct link between mutant SOD1 and a glial-derived toxic mediator.     

Simultaneous measurement of D-serine dehydratase and d-amino acid oxidase activities by the detection of 2-oxo-acid formation with reverse-phase high-performance liquid chromatography

N-methyl-D-aspartate receptors (NMDARs) play critical roles in excitatory synaptic transmission in the vertebrate central nervous system. NMDARs need D-serine for their channel activities in various brain regions. In mammalian brains, D-serine is produced from L-serine by serine racemase and degraded by D-amino acid oxidase (DAO) to 3-hydroxypyruvate. In avian organs, such as the kidney, in addition to DAO, D-serine is also degraded to pyruvate by D-serine dehydratase (DSD). To examine the roles of these two enzymes in avian brains, we developed a method to simultaneously measure DAO and DSD activities. First, the keto acids produced from D-serine were derivatized with 3-methyl-2-benzothiazolinone hydrazone to stable azines. Second, the azine derivatives were quantified by means of reverse-phase high-performance liquid chromatography using 2-oxoglutarate as an internal standard. This method allowed the simultaneous detection of DAO and DSD activities as low as 100 pmol/min/mg protein. Chicken brain showed only DSD activities (0.4+/-0.2 nmol/min/mg protein) whereas rat brain exhibited only DAO activities (0.7+/-0.1 nmol/min/mg protein). This result strongly suggests that DSD plays the same role in avian brains, as DAO plays in mammalian brains. The present method is applicable to other keto acids producing enzymes with minor modifications.     

Effect of Antipsychotic Drugs on the Gene Expression of NMDA Receptor Subunits in Rats

It has been hypothesized that glutamatergic neurons play an important role in clinical manifestations of schizophrenia and that the therapeutic effect of antipsychotic drugs is related to glutamatergic neurotransmission. To elucidate the effect of antipsychotic drugs on glutamatergic transmission, we examined gene expressions of NMDA receptor subunits Rl, R2A, R2B and R2C in the whole brains of rats after acute and chronic administrations of haloperidol and sulphide, using the Northern blot technique. The levels of NMDAR2B mRNAs decreased after the acute administration of haloperidol, but showed no change after the chronic administration. The levels of NMDAR2A and R2B mRNAs decreased after the acute administration of sulpiride, whereas the levels of R2A and R2B increased following the chronic administration. Neither haloperidol nor sulpiride influenced NMDAR1 mRNA levels. These data support differential expression of NMDA receptor subunits in rats upon treatment with haloperidol and sulpiride. The results imply that NMDAR2 subunits may be crucial in the regulation and modification of antipsychotic drugs.     

Molecular mechanisms underlying glutamatergic dysfunction in schizophrenia: therapeutic implications

Early models for the etiology of schizophrenia focused on dopamine neurotransmission because of the powerful anti-psychotic action of dopamine antagonists. Nevertheless, recent evidence increasingly supports a primarily glutamatergic dysfunction in this condition, where dopaminergic disbalance is a secondary effect. A current model for the pathophysiology of schizophrenia involves a dysfunctional mechanism by which the NMDA receptor (NMDAR) hypofunction leads to a dysregulation of GABA fast- spiking interneurons, consequently disinhibiting pyramidal glutamatergic output and disturbing the signal-to-noise ratio. This mechanism might explain better than other models some cognitive deficits observed in this disease, as well as the dopaminergic alterations and therapeutic effect of anti-psychotics. Although the modulation of glutamate activity has, in principle, great therapeutic potential, a side effect of NMDAR overactivation is neurotoxicity, which accelerates neuropathological alterations in this illness. We propose that metabotropic glutamate receptors can have a modulatory effect over the NMDAR and regulate excitotoxity mechanisms. Therefore, in our view metabotropic glutamate receptors constitute a highly promising target for future drug treatment in this disease.     

Alterations in NMDA receptor subunit levels in the brain regions of rats chronically administered typical or atypical antipsychotic drugs

It has been hypothesized that glutamatergic neurotransmission is related to the therapeutic effect of antipsychotic drugs. To test this hypothesis, we measured by use of the Western blot technique the polypeptide levels of NMDA receptor subunits, that is, NMDAR1, 2A, 2B, and 2C, in several regions of the rat brain after chronic treatment with haloperidol (HPD) or clozapine (CLZ). Each rat was intraperitoneally injected with HPD or CLZ at 10:00 h daily for 14 days. The brain regions examined were frontal cortex, striatum, nucleus accumbens, hippocampus, and cerebellum. Decreases in the polypeptide level of NMDAR2B were seen in hippocampus (but not in other brain regions) following the treatment with HPD or CLZ. Altered levels in NMDAR1-, 2A-, and 2C were not detected in any brain regions examined. We infer that an alteration in NMDAR2B in hippocampus is related to therapeutic effects of antipsychotic drugs.     

The N-methyl D-aspartate receptor glycine site and D-serine metabolism: an evolutionary perspective

The N-methyl D-aspartate (NMDA) type of glutamate receptor requires two distinct agonists to operate. Glycine is assumed to be the endogenous ligand for the NMDA receptor glycine site, but this notion has been challenged by the discovery of high levels of endogenous d-serine in the mammalian forebrain. I have outlined an evolutionary framework for the appearance of a glycine site in animals and the metabolic events leading to high levels of D-serine in brain. Sequence alignments of the glycine-binding regions, along with the scant experimental data available, suggest that the properties of invertebrate NMDA receptor glycine sites are probably different from those in vertebrates. The synthesis of D-serine in brain is due to a pyridoxal-5'-phosphate (B(6))-requiring serine racemase in glia. Although it remains unknown when serine racemase first evolved, data concerning the evolution of B(6) enzymes, along with the known occurrences of serine racemases in animals, point to D-serine synthesis arising around the divergence time of arthropods. D-Serine catabolism occurs via the ancient peroxisomal enzyme d-amino acid oxidase (DAO), whose ontogenetic expression in the hindbrain of mammals is delayed until the postnatal period and absent from the forebrain. The phylogeny of D-serine metabolism has relevance to our understanding of brain ontogeny, schizophrenia and neurotransmitter dynamics.     

Immunocytochemical Characterization of Glutamate Dehydrogenase in the Cerebellum of the Rat

The immunocytochemical distribution of glutamate dehydrogenase was studied in the cerebellum of the rat using antibodies made in rabbit and guinea pig against antigen purified from bovine liver. Antiserum was found to block partially enzymatic activity both of the purified enzyme and of extracts of the rat cerebellum. Using immunoblots of proteins of rat cerebellum, a major immunoreactive protein and several minor immunoreactive proteins were detected with antiserum. Only a single immunoreactive protein was detected using affinity-purified antibody preparations. This protein migrates with a molecular weight identical to that of the subunit of glutamate dehydrogenase. Further evidence that the antibodies were selective for glutamate dehydrogenase in rat cerebellum was obtained through peptide mapping. Purified glutamate dehydrogenase and the immunoreactive protein from rat cerebellum generated similar patterns of immunoreactive peptides. No significant cross-reaction was observed with glutamine synthetase. Immunocytochemistry was done on cryostat- and Vibratome-cut sections of the cerebellum of rats that had been perfused with cold 4% paraformaldehyde. Glial cells were found to be the most immunoreactive structures throughout the cerebellum. Most apparent was the intense labeling of Bergmann glial cell bodies and fibers. In the granule cell layer, heavy labeling of astrocytes was seen. Purkinje and granule cell bodies were only lightly immunoreactive, whereas stellate, basket, and Golgi cells were unlabeled. Labeling of presynaptic terminals was not apparent. These findings suggest that glutamate dehydrogenase, like glutamine synthetase, is enriched in glia relative to neurons.     

D-amino acids in the brain: the biochemistry of brain serine racemase

It has been recently established that in various brain regions D-serine, the product of serine racemase, occupies the so-called 'glycine site' within N-methyl D-aspartate receptors. Mammalian brain serine racemase is a pyridoxal-5' phosphate-containing enzyme that catalyzes the racemization of L-serine to D-serine. It has also been shown to catalyze the alpha,beta-elimination of water from L-serine or D-serine to form pyruvate and ammonia. Serine racemase is included within the group of type II-fold pyridoxal-5' phosphate enzymes, together with many other racemases and dehydratases. Serine racemase was first purified from rat brain homogenates and later recombinantly expressed in mammalian and insect cells as well as in Escherichia coli. It has been shown that serine racemase is activated by divalent cations like calcium, magnesium and manganese, as well as by nucleotides like ATP, ADP or GTP. In turn, serine racemase is also strongly inhibited by reagents that react with free sulfhydryl groups such as glutathione. Several yeast two-hybrid screens for interaction partners identified the proteins glutamate receptor interacting protein, protein interacting with C kinase 1 and Golga3 to bind to serine racemase, having different effects on its catalytic activity or stability. In addition, it has also been proposed that serine racemase is regulated by phosphorylation. Thus, d-serine production in the brain is tightly regulated by various factors pointing at its physiologic importance. In this minireview, we will focus on the regulation of brain serine racemase and d-serine synthesis by the factors mentioned above.     

Chlorpromazine oligomer is a potentially active substance that inhibits human D-amino acid oxidase, product of a susceptibility gene for schizophrenia

D-amino acid oxidase (DAO), a potential risk factor for schizophrenia, has been proposed to be involved in the decreased glutamatergic neurotransmission in schizophrenia. Here we show the inhibitory effect of an antipsychotic drug, chlorpromazine, on human DAO, which is consistent with previous reports using porcine DAO, although human DAO was inhibited to a lesser degree (K(i) = 0.7 mM) than porcine DAO. Since chlorpromazine is known to induce phototoxic or photoallergic reactions and also to be transformed into various metabolites, we examined the effects of white light-irradiated chlorpromazine on the enzymatic activity. Analytical methods including high-resolution mass spectrometry revealed that irradiation triggered the oligomerization of chlorpromazine molecules. The oligomerized chlorpromazine showed a mixed type inhibition with inhibition constants of low micromolar range, indicative of enhanced inhibition. Taken together, these results suggest that oligomerized chlorpromazine could act as an active substance that might contribute to the therapeutic effects of this drug.